US7632674B2 - Apparatus for stimulating an animal cell and recording its physiological signal and production and use methods thereof - Google Patents

Apparatus for stimulating an animal cell and recording its physiological signal and production and use methods thereof Download PDF

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US7632674B2
US7632674B2 US10/499,098 US49909804A US7632674B2 US 7632674 B2 US7632674 B2 US 7632674B2 US 49909804 A US49909804 A US 49909804A US 7632674 B2 US7632674 B2 US 7632674B2
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conductive polymer
stimulating
recording
microelectrodes
animal cell
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US20050106708A1 (en
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Wanli Xing
Zhe Yu
Guangxin Xiang
Liangbin Pan
Jing Cheng
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Tsinghua University
CapitalBio Corp
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Tsinghua University
Capital Biochip Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to an apparatus for stimulating, particularly electrically stimulating, an animal cell and recording its physiological signals, as well as methods of making and using such apparatus.
  • the ability of controlling neuronal growth and differentiation will be useful for the study of the underlying mechanisms of the nervous system, the treatment of neuronal diseases, and the effective repair of damages to nerve tissues. It has therefore been the focus of scientific research for a long time.
  • Conductive polymers represent a new class of materials whose electrical and optical properties can be controllably varied over a wide range, often in a reversible manner. Conductive polymers are stable, can be used in physiological cell culture media or body fluid for extended time, and have good compatibility with neurons.
  • microelectrodes or arrays of microelectrodes to record electrophysiological signals of neurons and nerve networks has been studied since the 1970's, and tremendous progress has been made in recent years.
  • One way is to insert multiple microelectrodes into a live subject to measure extracellular signals.
  • the electrodes can be in the shape of spikes, with multiple electrodes being a cluster of spikes (E. Fernandez, J. M. Ferrandez, J. Ammermuller, R. A. Normann, Population coding in spike trains of simultaneously recorded retinal ganglion cells, Brain Research, 2000, 887, 222-229; D. J. Warren, E. Fernandez, R. A.
  • Jimbo Activity-dependent enhancement in the reliability of correlated spike timings in cultured cortical neurons, Biological Cybernetics, 1999, 80, 45-55; M. P. Maher, J. Pine, J. Wright, Y. C. Tai, The neurochip: a new multielectrode device for stimulating and recording from cultured neuron, Journal of Neuroscience Methods, 1999, 87, 45-56); the third method is to make spikes containing the microelectrode arrays of the second method along with embryonic neurons, insert the spikes into a live subject, and observe the integration and communication of signals between cells on the microelectrode array and cells in the subject. (J. Pine, M. Maher, S. Potter, Y. C.
  • the purpose of the present invention is to provide an efficient, convenient, and accurate apparatus for stimulating an animal cell and recording its physiological signal.
  • the present invention provides an apparatus for stimulating an animal cell and recording its physiological signal, such apparatus comprising a substrate comprising a poor conductive material, wherein on at least one face of the substrate is provided at least one unit conductive polymer layer and at least one good conductive microelectrode.
  • an intermediate layer between the substrate and the conductive polymer layer can be provided.
  • Such intermediate layer can be made of gold or platinum.
  • One function of the intermediate layer is to serve as a carrier for electrical polymerization. Another function is to allow conductive polymer to adhere strongly.
  • the substrate can be a two-dimensional planar structure; or it can contain one or more wells for positioning cells.
  • the material used to make the substrate can be rigid or flexible, and can be chosen from silicon, glass, polymer, or metal oxides.
  • the conductive polymer serves to guide the outgrowth of the neurites. Accordingly, the conductive polymers can form a grid-like pattern with disconnected junctions, with nerve cells at the junctions. The neurites will therefore grow along the conductive polymer pattern and communicate with each other. Because the diameters of neurites are usually 1-2 microns, the width of each band of conductive polymers is usually smaller than 5 microns. If the band is too narrow or too broad, the ability of the conductive polymers to guide the neurites properly will be affected. The pattern of the conductive polymers should be chosen in a way that facilitates the formation of nerve networks among the neurites.
  • the conductive polymer described herein can comprise multiple units. These units can be interconnected and share a single pair of stimulating electrodes. Alternatively, the units may not be connected with each other, each unit having their own pair of stimulating electrodes. If the units are not interconnected, the stimulating parameters (such as current intensity, duration, amplitude, and frequency) for each conductive polymer unit can be either the same or different.
  • the thickness of the conductive polymer layer described herein can be uniform or not uniform.
  • the thickness of the conductive polymer layer can be between several nanometers to several millimeters.
  • the thickness of the conductive polymer layer is preferably below 500 nanometers. This provides high light permeability and allows the polymers to properly adhere to the substrate.
  • the material that the conductive polymer can be made of includes polyaniline, polypyrrole, polythiophene or their derivatives, copolymers, or mixtures thereof.
  • the conductivity of the conductive polymer can be adjusted according to spatial and temporal requirements. When microelectrodes are used to record electrophysiological signals, the conductive polymer layer can either be adjusted to be nonconductive or remain being conductive, as long as the measurement of the electrophysical signals is not affected.
  • microelectrodes described herein may comprise more than one microelectrodes, which can be arranged into microelectrode arrays.
  • the material from which the microelectrodes are made can be gold, platinum, or indium-tin oxide (ITO).
  • the conductive polymer layer and the microelectrodes described herein can be connected or not connected to each other. When not connected, the conductive polymer layer and microelectrodes can be about 1 to about 50 microns apart. The distance between conductive polymer and microelectrode in each unit can be the same or different.
  • the area above the substrate that is not covered by conductive polymers and is not the area for cell growth or microelectrode measurement can be coated with insulation materials.
  • the insulation material can be made of nonconductive materials (such as polyimide or other materials with good biocompatibility) that are not toxic to cells.
  • the thickness of the insulation layer is usually 5 to 100 microns.
  • Another purpose of the present invention is to provide a simple method of making the apparatus for stimulating animal cells and recording the physiological signals described above.
  • the method of making an apparatus for stimulating animal cells and recording the physiological signals comprises the steps of: (a) depositing good conductive microelectrodes and their connecting wires on a substrate through evaporation or sputter; (b) depositing at least one unit of conductive polymer layer with a desired pattern on the substrate by PVD, CVD, electrical polymerization, or macromolecule self-assembly; and (c) depositing some metallic wires underneath the conductive polymer layer for connecting the conductive polymers to the stimulating electrodes.
  • Another purpose of the present invention is to provide a method of using an apparatus for stimulating animal cells and recording physiological signals.
  • the method of using an apparatus for stimulating animal cells and recording physiological signals comprises the steps of: (a) passing electrical current through conductive polymers either continuously or intermittently, wherein the electrical stimulation can be direct current or alternating current, wherein the electric current intensity can be of pA to mA scale, and wherein for alternating current stimulation, the stimulation frequency can be 1-10 6 Hz; and (b) recording physiological signals generated from cells being tested through the microelectrodes by measuring electrical current, electrical potential, or impedance electrical signals.
  • the apparatus for stimulating animal cells and recording physiological signals described herein can be two dimensional squares, circles, or any other irregular shapes.
  • the apparatus is a three-dimensional hollow cylinder, a globe, a cubic, a cuboid, a cone, or any other irregular shapes.
  • FIG. 1 provides a structural diagram of a subregion of an apparatus of the present invention.
  • FIG. 2 provides an overview of an apparatus of the present invention.
  • FIG. 3 provides a magnified view of microelectrode arrays.
  • FIG. 4 provides a sectional view of a single culture unit used for studying nerve networks in vitro.
  • FIG. 5 provides a three dimensional diagram of an apparatus of the present invention that is used for implanting into a live subject.
  • An apparatus for stimulating animal cells and recording its physiological signals can be made using the above method.
  • the substrate can be chosen from a wide range of materials, as long as it is a poor conductive material.
  • the thickness and shape of the substrate can be chosen according to the needs of the experiment. To make efficient use of the space, more than one surface of the substrate can be utilized as described above.
  • the electrode layer can be made of platinum, ITO, or other kinds of good electrically conductive materials.
  • the thickness of the electrode layer is preferably between 30 nm to 400 nm.
  • the material from which the conductive polymers are made of can be chosen from a wide variety of materials, and depends on the needs of the experiment.
  • the material can be polyaniline, polypyrrole, polythiophene, or their derivatives, copolymers, or mixtures thereof. Other methods of coating or photoetching can also be used to generate electrodes and conductive polymer layers and to form desired patterns.
  • the apparatus for stimulating animal cells and recording its physiological signals comprises the 400 micron-thick square silicon substrate 4 ; the 100-micron thick polypyrrole conductive polymer layer 3 on top of substrate 4 , the conductive polymer layer being connected to two electrodes (not indicated in this figure); the sixteen 40 nm-thick platinum microelectrodes 2 on top of substrate 4 , each microelectrode having an output electrode (not indicated in this figure). There is a 25-micron gap ( 8 ) between the conductive polymer 3 and the microelectrode 2 .
  • a polyimide insulation layer 1 Areas on substrate 4 that are not covered by conductive polymers 3 and are not areas for cell growth ( 10 ) or microelectrodes measurement are covered by a polyimide insulation layer 1 .
  • the insulation layer has a thickness of 10 microns, forming a 3-7 micron-wide channel ( 9 ) that prevents cells from escaping.
  • the chamber is made of biocompatible materials such as polyimide.
  • the height of the culture chamber 5 is usually bigger than 100 mm, and the area of the chamber depends on the number and distribution of the microelectrodes in the microelectrode array.
  • the size and shape of the apparatus, as well as the choice of the surface or surfaces of the substrate for depositing conductive polymer layers or microelectrodes, are determined by the needs of the experiment.
  • the two lateral sides of the substrate 4 are both covered with conductive polymer layer 3 and microelectrodes 2 . Neurons can be placed on both sides of the substrate, thus to increase cell density and enhance efficiency.
  • the apparatus for implanting in vivo contains a top layer 6 . This layer is useful for preventing cell damages during implanting and preventing cells from escaping in the live subject.
  • the material from which layer 6 is made of is preferably biocompatible, stable, and nonconductive (such as polyimide).
  • the electrical stimulation can be direct current or alternating current, and the stimulation frequency can be 1-10 6 Hz.
  • the physiological signals of the cell being studied are measured through microelectrode 2 .
  • the growth of the stimulated cells is measured by parameters such as the size of the cell body, the length of the neurites, is measured and compared with those of unstimulated cells.
  • cells are stimulated by electrical or chemical signals, and the response of the cells to the various stimulations is monitored through the microelectrodes, by measuring the changes of the electrical potential on the cell membrane.
  • the neuronal network on the apparatus is used to connect damaged nerve fibers in the subject.
  • the apparatus is also used to measure the response of neurons in local nerve tissues to external stimuli (including electrical and medicinal stimuli). Furthermore, the apparatus can be used to electrically stimulate local nerve tissues, thereby stimulate the repair and regeneration of damaged nerves.
  • the present invention applies conductive polymer and microelectrode fabrication technologies to neural science, and utilizes electrical stimulation through conductive polymers to effectively control the speed and direction of neurite outgrowth. This allows effective recording of electrophysiological signals of nerve network in vitro, and makes it possible to further study signal transduction and underlying mechanisms of the nervous system.
  • the present invention also provides an implantable apparatus that allows the implantation of nerve cells in vivo, integration of implanted nerve cells with the main nerve system, and repair of damaged nerve tissues. The apparatus therefore has important medical application values. It is also expected that the apparatus will be useful for the treatment of urine incontinence, retina injury, or other neuronal diseases.
  • the apparatus of the present invention is not only useful for the study of different kinds of cells derived from various animal nervous systems, but also useful for the study of cells derived from non-neuronal cells.

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US10/499,098 2001-12-17 2002-11-29 Apparatus for stimulating an animal cell and recording its physiological signal and production and use methods thereof Expired - Fee Related US7632674B2 (en)

Applications Claiming Priority (3)

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CNB011403381A CN100344960C (zh) 2001-12-17 2001-12-17 刺激动物细胞并记录其生理信号的装置及其生产使用方法
CN01140338.1 2001-12-17
PCT/CN2002/000856 WO2003056321A1 (fr) 2001-12-17 2002-11-29 Dispositif pour stimuler une cellule animale et enregistrer son signal physiologique, procedes de production et d'utilisation correspondants

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EP (1) EP1464952B1 (de)
JP (1) JP2005514019A (de)
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AT (1) ATE365913T1 (de)
AU (1) AU2002354331A1 (de)
DE (1) DE60220947T2 (de)
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US20110208029A1 (en) * 2007-10-22 2011-08-25 Centre National De La Recherche Scienfifique (Cnrs) Device for stimulating living tissue by microelectrodes and removable module and use thereof
US8352045B2 (en) * 2007-10-22 2013-01-08 Centre National De La Recherche Scientifique (Cnrs) Device for stimulating living tissue by microelectrodes and removable module and use thereof

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US20050106708A1 (en) 2005-05-19
EP1464952B1 (de) 2007-06-27
AU2002354331A1 (en) 2003-07-15
JP2005514019A (ja) 2005-05-19
CN100344960C (zh) 2007-10-24
EP1464952A4 (de) 2005-11-30
ATE365913T1 (de) 2007-07-15
DE60220947T2 (de) 2008-02-28

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